Fungal L-asparaginase: Strategies for production and food applications

Food Research International 126 (2019) 108658

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Review

Fungal L-asparaginase: Strategies for production and food applications a,⁎

a

T b

Marília Crivelari da Cunha , Jessika Gonçalves dos Santos Aguilar , Ricardo Rodrigues de Melo , Sheila Tiemi Nagamatsuc, Faraat Alid, Ruann Janser Soares de Castroa, Hélia Harumi Satoa a

Department of Food Science, School of Food Engineering, University of Campinas, Campinas, SP, Brazil Brazilian Bioethanol Science and Technology Laboratory (CTBE), Brazilian Center for Research in Energy and Materials (CNPEM), Campinas, SP, Brazil Departament of Genetics, Evolution, Microbiology and Imunology, Institute of Biology, University of Campinas, Campinas, SP, Brazil d Pharmaceutical Chemistry Division, Indian Pharmacopoeia Commission, Ministry of Health & Family Welfare, Government of India, Sector-23, Rajnagar, Ghaziabad, Uttar Pradesh 201002, India b c

A B S T R A C T

L-asparaginase (L-asparagine amidohydrolase EC 3.5.1.1) is of great importance in pharmaceutical and food applications. This review aims to describe the production and use of fungal L-asparaginase focusing on its potential as an effective reducer of acrylamide in different food applications. Fungal asparaginases have been used as food additives and have gained importance due to some technical advantages, for example, fungi can grow using low-cost culture mediums, and the enzyme is extracellular, which facilitates purification steps. Research aimed at the discovery of new L-asparaginases, mainly those produced by fungi, have great potential to obtain cheaper enzymes with desirable properties for application in food aiming at the reduction of acrylamide.

1. Introduction Acrylamide is known to be a neurotoxic, genotoxic, and carcinogenic substance in animal tests (Pennisi et al., 2013) and it was classified as a probable human carcinogen (IARC, 1994). This substance is formed by the Maillard reaction in food containing L-asparagine and reducing sugars, such as glucose and fructose, heated at high temperatures and low humidity. This reaction also forms desirable compounds of color, flavor, and aroma (Thomas & Thomas, 2014) in cooked or fried food. According to the European Food Safety Authority, the most important food groups contributing to exposure to acrylamide are French fries, bakery products, and coffee (EFSA, 2015). To mitigate acrylamide levels in bread, biscuits, potato chips, coffee, and others food containing L-asparagine and reducing sugars, the food industry faces the challenges of changing pre-set process parameters without compromising the texture, taste, or appearance of its products (Friedman, 2015). The use of the enzyme L-asparaginase presents high technological potential to reduce the acrylamide content in thermally treated food without compromising their sensorial or nutritional aspects (Hendriksen, Kornbrust, Østergaard, & Stringer, 2009). L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) is an enzyme capable of catalyzing the hydrolysis of L-asparagine into L-aspartic acid and ammonia, thereby reducing the content of L-asparagine, the precursor amino acid of this toxic compound (Capuano & Fogliano, 2011). L-asparaginases are widely distributed in plants, animals, and microorganisms (Batool, Makky, Jalal, & Yusoff, 2016; Zuo, Zhang, Jiang,

⁎

& Mu, 2015). However, the most important source of L-asparaginase is microorganisms (Lopes, Oliveira-Nascimento, Ribeiro, Breyer, et al., 2017). To treat of some types of leukemia and lymphoma, bacterial Lasparaginase is used as an injectable drug (Batool et al., 2016), particularly in children with acute lymphoblastic leukemia (Izadpanah, Homaei, Fernandes, & Javadpour, 2018). For pharmaceutical uses, Lasparaginase is obtained from Escherichia coli and Erwinia carotovora (also known as Erwinia chrysanthemi) (Cachumba et al., 2016). However, the bacterial source of this enzyme may cause allergic reactions (Izadpanah et al., 2018). Therefore, it is necessary to search for other microbial sources of L-asparaginase, such as eukaryotic microorganisms, thus obtaining an enzyme with fewer adverse effects (Patro & Gupta, 2012). Currently, fungal L-asparaginases are considered safe by JECFA (2007) and are used as food additives (Xu, Oruna-Concha, & Elmore, 2016). However, the yield of L-asparaginase produced is not enough to meet the demand (Jha et al., 2012) creating the need for new techniques to increase the yield, such as the use of statistical tools. For enzyme production, other fermentation methods have been adopted, such as the solid-state fermentation, since it has more advantages than the submerged fermentation, industrially used (Thomas, Larroche, & Pandey, 2013). In the present review, fungal sources of L-asparaginase are discussed: general aspects, methods for enzyme production, purification, and applications in food.

Corresponding author. E-mail addresses: [email protected] (M.C. da Cunha), [email protected] (R.J.S. de Castro), [email protected] (H.H. Sato).

https://doi.org/10.1016/j.foodres.2019.108658 Received 24 March 2019; Received in revised form 30 August 2019; Accepted 6 September 2019 Available online 09 September 2019 0963-9969/ © 2019 Elsevier Ltd. All rights reserved.

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Mohapatra, & Gupta, 2014; Shrivastava, Khan, Shrivastav, Jain, & Singhal, 2012) can be highlighted. Some genera, however, may produce mycotoxins, among them aflatoxin, ochratoxin, zearalenone, among others. Mycotoxins are secondary metabolites that have toxic effects for humans (Maziero & Bersot, 2010). Fungal L-asparaginases used in the food industry are considered GRAS (Generally Recognized as Safe) and are obtained from A. oryzae and A. niger. Commercially, PreventASe® (DSM) and Acrylaway® (Novozymes) preparations are currently available for the reduction of acrylamide in the food industry. PreventASe was obtained by Aspergillus niger, presenting an optimal pH between 4 and 5 and an optimum temperature of 50 °C (Xu et al., 2016). PreventASe® reduces the formation of acrylamide in high pH (up to 9) applications such as corn chips, biscuits, and crackers (DSM, 2018). The company Novozymes S/A developed the production of the Lasparaginase based on the cloning of Aspergillus oryzae. L-asparaginase from A. oryzae presents an optimum pH between 6 and 7, with good activity in the range between pH 5 and 8 and optimum temperature of 60 °C (Pedreschi, Kaack, & Granby, 2008). These products can be used as co-adjuvants of the technology and are currently used in several countries, such as the United States, Australia, New Zealand, China, Russia, Mexico, and several European countries (Xu et al., 2016). However, food composition may influence the action of enzymes (Xu et al., 2016). Therefore, the ideal L-asparaginase enzyme for food use should be stable during food processing and proteolysis and not induce allergic or toxic effects after consumption (Friedman, 2015).

Fig. 1. Distribution of L-asparaginase according to the National Biotechnology Information Center (NCBI).

2. Fungal L-asparaginase

2.2. Production of L-asparaginase by fungi

2.1. General aspects

Different methods are used for production of L-asparaginase, such as solid-state fermentation (SSF) and submerged fermentation (SF) using different microorganisms (Batool et al., 2016). The SF processes are well established and provide good yield, but generate large amounts of wastewater and have a high production cost (Izadpanah et al., 2018). On the other hand, SSF is an attractive alternative for SF and has great potential for enzyme production. SSF has achieved great relevance in bioprocesses, as it uses low cost agroindustrial waste as a substrate and offers potential environmental benefits (Thomas et al., 2013). In addition, this type of fermentation resembles the natural habitat of microorganisms and is well adapted to the metabolism of fungi (Singhania, Patel, Soccol, & Pandey, 2009). There are several important factors that have a huge impact on SSF success. These factors include pH, temperature, aeration, water and moisture activity, origin of the solid substrate, and particle size and shape. Among several critical factors, the moisture and origin of the solid substrate are the most important factors affecting SSF processes. The selection of moisture depends on the microorganism employed and on the origin of the substrate. The fungi need low humidity between 40 and 60%, however, the selection of the substrate depends on several factors, mainly related to availability and cost (Singhania et al., 2009). Solid substrates, mainly residues or agricultural by-products, are being used as substrates for fungal L-asparaginase production (Dias et al., 2015; Kumar & Manonmani, 2013; Meghavarnam & Janakiraman, 2017; Mishra, 2006). These substrates act as physical support and source of nutrients for the production of enzymes. Therefore, for the commercial production of the enzyme the selection of an appropriate substrate is an important step (Meghavarnam & Janakiraman, 2017). Table 1 shows different substrates, the parameters used in the solid-state fermentation, and the maximum activity of Lasparaginase by the fungi. The industrial production of bacterial and fungal L-asparaginase worldwide is mainly performed by SF (Kumar & Manonmani, 2013). Lasparaginase production is strongly influenced by the composition of the fermentation medium, especially carbon and nitrogen sources, and physical factors such as temperature, pH, agitation, inoculum concentration, and fermentation time (Baskar et al., 2010; Hymavathi,

L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) can be found from microorganisms (bacteria, fungi, yeasts, actinomycetes and algae) to higher organisms (plants, vertebrates and animal tissues) (Zuo et al., 2015). According to the National Center for Biotechnology Information (NCBI), L-asparaginase sequences are mainly distributed in the bacterial kingdom, accounting for 95.5% of deposited protein sequences (221,303 of 231,770 protein sequences). However, L-asparaginase can also be found in the fungi (1.68%), animal (1.25%), plant (0.24%), archaea (0.88%), and virus (< 0.01%) kingdoms (Fig. 1). The search for other sources of L-asparaginase, for instance eukaryotic microorganisms, becomes important. Eukaryotic microorganisms such as yeasts and filamentous fungi have potential for the production of L-asparaginase (Baskar, Sriharini, Sripriya, & Renganathan, 2010). The L-asparaginases sequences from kingdom fungi are divided into six subclasses: ascomycetes (85.2%), basidiomycetes (11.1%), chytrids, glomeromycetes, microsporidians, and blastocladiomycetes (< 1% each). From all fungi database, 212 sequences were used to construct the phylogenetic tree (Fig. 2) with sequences that aligned to an Aspergillus niger L-asparaginase. The distribution shifts the percentage of sequences by subclasse basidiomycetes to ascomycetes, with 11 basidiomycetes (5.1%), and 199 ascomycetes (divided into ascomycetes, budding yeasts and fission yeasts). This difference can be explained by an increase in the proportion of well annotated L-asparaginase in budding yeasts strains, mainly of the genus Candida which represent 12.7% of the proteins used in phylogeny. Microorganisms are considered the most important source of L-asparaginase, since the anti-tumor L-asparaginase activity of Escherichia coli (Mashburn & Wriston Jr., 1964) was first reported. Microorganisms can produce several types of L-asparaginase that differ in their location (intracellular and extracellular) and properties (Zuo et al., 2015). Fungal L-asparaginases have become important because they are excreted in the extracellular environment, are easy to extract, and process downstream (Batool et al., 2016; El-Naggar, El-Ewasy, & El-Shweihy, 2014). Among the fungi producing L-asparaginase, the genera Aspergillus, Penicillium, Fusarium, Trichoderma, and Cladosporium (Kumar & Manonmani, 2013; Lincoln, Niyonzima, & More, 2015; Patro, Basak, 2

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Fig. 2. Phylogenetic distribution of L-asparaginase in kingdom fungi. In the phylogenetic tree construction, 3915 protein sequences were blasted against an Aspergillus niger L-asparaginase, being keeping all sequences with p-value alignment > 1.e-05. Then, were filtered to eliminate hypothetical proteins, and maintain only the highest sequence for each strain. The sequence of E.coli (L-asparaginase 1) was applied as an external group to those 212 sequences remained. All sequences were aligned with MAFFT (version 7 - https://mafft.cbrc.jp/alignment/server/) using auto strategy. To phylogeny tree was used Neighbor joining method to all gap-free sites (30AAs) and 1000 bootstraps in MAFFT server. The Tree was design in iTOL software (https://itol.embl.de/).

temperature, a range between 30 and 37 °C for the optimum production of this enzyme was reported for fungal species (Dange & Penshwe, 2011; Elshafei, Hassan, Abouzeid, Mahmoud, & Elghonemy, 2012; Kumar & Manonmani, 2013; Thakur, Lincoln, Niyonzima, & More, 2014). Most studies have shown that using agitation around 160 rpm and 4 or 5 days of incubation, at temperatures of 30 °C, the production of fungal L-asparaginase is easily observed (Souza et al., 2017). The evaluation of nutritional requirements and physical conditions is an important step in the development of bioprocesses (Baskar & Renganathan, 2011). Currently, optimization methods are used to make a bioprocess economically viable. Some statistical methods of experimental planning have been used to optimize the process of L-asparaginase production from fungi (Souza et al., 2017). The traditional technique of one factor at a time used to optimize a multivariate system leads to errors in interpretation of results, since it neglects the effects of interactions among factors (El-Naggar et al., 2014). The use of experimental planning eliminates the disadvantages of using the one factor at a time technique (El-Naggar et al., 2014). This optimization process involves three main steps: (i) performing the experiments using a statistically designed matrix, (ii) estimating the coefficients in a mathematical model, predicting their response, and (iii) verifying the suitability of the model (Kumar & Manonmani, 2013). This technique has several advantages that include requiring a smaller number of experiments, application in experiments with several factors, besides considering the interaction between these, and to generate a mathematical model, facilitating the discovery of the most adequate condition and the prediction of response (El-Naggar et al., 2014). Statistical tools such as Plackett-Burman planning and surface response methodology have been used in several optimization studies. Another statistical tool, artificial neural network coupled to the genetic algorithm, has also been used in optimization studies in bioprocesses (Baskar & Renganathan, 2012). In addition, other types of statistical methods have been used to optimize L-asparaginase production by fungi, such as Simplex Mixture design (Dias et al., 2015), Box-Behnken design (Uppuluri, Dasari, Sajja, Jacob, & Reddy, 2013), and Latin Square design (Baskar et al., 2010). Baskar and Renganathan (2012) used the strategy of sequential

Sathist, Rao, & Prakasham, 2009; Souza et al., 2017). Each organism has its own special conditions for the maximum production of enzymes, so the optimization of average components and culture parameters is essential in the biological process (Souza et al., 2017). Table 2 shows different subtrates for culture medium and the parameters used in the submerged fermentation and the maximum activity of L-asparaginase by the fungi. Different substrates for culture medium have been exploited to produce L-asparaginase. However, the most important components in the fermentation medium are the carbon source and the nitrogen source (Cachumba et al., 2016). The influence of various carbon sources such as glucose, sucrose, fructose, lactose, maltose, and starch have been studied and several studies have suggested that glucose is the best carbon source to produce this enzyme (Doriya & Kumar, 2016). However, it is known that the synthesis of L-asparaginase requires low concentration of carbon sources since it is under catabolic repression (Mukherjee, Majumdar, & Scheper, 2000). Some studies suggest that, in the case of L-asparaginase biosynthesis, the depressive effect of carbohydrates may be a function of the ability to lower pH of the fermentation medium (Heinemann & Howard, 1969; Mukherjee et al., 2000). Nitrogen is an important nutrient for microorganisms (Stanbury, Whitaker, & Hall, 1995). Several studies have shown that the best nitrogen sources to achieve high yields of L-asparaginase are L-asparagine (Doriya & Kumar, 2016; Farag, Hassan, Beltagy, & El-Shenawy, 2015), and L-proline (Sarquis, Oliveira, Santos, & Costa, 2004). In addition to these two amino acids, other nitrogen sources were also considered important for the production of L-asparaginase, such as urea (Baskar et al., 2010), yeast extract, and peptone (Baskar & Renganathan, 2010). Although L-asparagine is an inducer in the production of L-asparaginase, this amino acid can also be considered a limiting factor for the production of this enzyme (Baskar & Renganathan, 2011). The pH of the fermentation medium plays a vital role in the transport of several nutrients through the cell membrane and in the increase of L-asparaginase production (Farag et al., 2015). In most studies on fungal L-asparaginase production, culture medium in the range of pH 6.3 to 9.0 was used for optimum enzyme production (Kumar & Manonmani, 2013; Dange & Penshwe, 2011). In relation to the 3

Soybean bran Passion fruit peel meal Mixture of cotton seed cake (2/3), wheat bran (1/6), and red gram husk (1/6) Wheat bran Sesame (black) oil cake Mixture of wheat bran (1/3), soybean meal (1/3) and cottonseed meal (1/3) Mixture of rice husk and wheat bran (3:2) Wheat bran Soybean meal Soybean meal

Aspergillus niger Aspergillus niger LBA 02 Aspergillus sp. Aspergillus fumigatus WL002 Aspergillus niger C4 Aspergillus niger LBA 02 Trichoderma viride Cladosporium Fusarium equiseti Fusarium culmorum (ASP-87)

6.5 – 8 5 6.5 – 5 5.8 7 7

pH 30 ± 2 30 35 37 29.31 30 28 30 45 30

Temp. (°C)

4

Trichoderma viride sp.

Modified CzapeckDox Modified CzapeckDox

Czapeck-Dox Modified CzapeckDox Modified CzapeckDox Modified CzapeckDox Czapeck-Dox

Aspergillus aculeatus Aspergillus oryzae CCT 3940

Aspergillus terreus MTCC 1782 Aspergillus terreus MTCC 1782 Aspergillus terreus MTCC 1782 Mucor hiemalis

Medium

Microorganism

5.85

1.8% L-asparagine

0.6% maltose

0.4% glucose 0.5% peptone

1.25% L-asparagine

6.2

1% urea

6.5

7

6.2

1.5% corn flour and 0.2% glucose 0.64% glucose

0.2% glucose

6.2 8

pH

1% L-asparagine 2% L-proline, 0.2% L-asparagine and 0.5% yeast extract 1% L-asparagine

Nitrogen source (w/v)

0.2% glucose 0.5% glucose

Carbon source (w/v)

37

30

32.08

30

30

30 30

Temp. (°C)

Table 2 Types of culture media, C and N sources, parameters used in submerged fermentation and maximum L-asparaginase activity.

Solid substrate

Microorganism

Table 1 Types of solid substrates, parameters used in solid state fermentation and maximum L-asparaginase activity.

1% spore suspension 5 mm disc of inoculum 5 mm disc of inoculum

123.5 rpm – –

160 rpm

~650 U/mL

1203.55 U

38.57 U/mL

33.25 U/mL

5 × 107 spores 107 to 108 spores/mL

33.59 U/mL

1% spore suspension 3 × 107 spores

– 150 rpm 180 rpm

380.14 IU/mL 67.49 U/mL

Spore concentration

Lincoln et al. (2015)

Baskar and Renganathan (2011) Thakur et al. (2014)

Baskar et al. (2010)

Doriya and Kumar (2016)

Dange and Penshwe (2011) Dias and Sato (2016)

Reference

Mishra (2006) Cunha, Silva, Sato, and Castro (2018) Dorya and Kumar (2018) Dutta et al. (2015) Uppuluri et al. (2013) Dias et al. (2015) Elshafei and El-Ghonemy (2015) Kumar and Manonmani (2013) Kaliwal and Hosamani (2011) Meghavarnam and Janakiraman (2017)

Reference

L-asparaginase activity

40.9 ± 3.35 U/g 3746.78 U/gds 12.57 U/mL 360 IU/mg of protein 355.88 U/gds 89.22 U/g 71.87 ± 3.19 U/gds 3.74 U 3.26 IU 7.21 U/gds

L-asparaginase activity

Agitation rate

70 60 70 90 96.02 50 75 58 70 70

Moisture (%)

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Ammonium sulfate precipitation, Dialysis, G-25 column, G-150 column Ultrafiltration (30 kDa), Ammonium sulfate precipitation (60%), DEAE-Sepharose column, Sephadex G-100 Etanol precipitation, DEAE cellulose

Aspergillus aculeatus

Methanol precipitation, DEAE-cellulose column, Sepharose 6B Acetone precipitation, Affinity chromatography with lectin-agarose Heat treatment for 20 min at 50 °C, Sephadex G-100 and G-200 Ammonium sulfate precipitation, G-100 column

Cladosporium sp.

5

Ammonium sulfate precipitation, Sephadex G100–120, DEAE-cellulose column Nickel-iminodiacetic acid column

Acetone precipitation, DEAE-cellulose column

Rhizomucor miehei

Trichoderma viride

Penicillium sp.

Penicillium brevicompactum NRC 829 Penicillium digitatum

Mucor hiemalis

Aspergillus sp. ALAA-2000

Ammonium sulfate precipitation (80%), dialysis, QSepharose Fast Flow, SP Sepharose Fast Flow, CM Sepharose Fast Flow Ammonium sulfate precipitation, Sephadex G-200

Aspergillus oryzae CCT 3940

Aspergillus niger AKV-MKBU

Aspergillus fumigatus WL002

Method of purification

Microorganism

Table 3 Purification and properties of some fungal L-asparaginases.

78.2

1984.8

13.0

2.6

1.9

60.94

833.15 13.97

151.12

4.59

867.7

574.24

69.43

83.3

8.3

28.6

282

0.4

10.36

232.04

355.03

46.75

267.75

Purification fold

207

Specific activity (IU/mg of protein)

99 ± 1 kDa

133.7 kDa

7

7

7

7

– 66 kDa

8

7

6 (AYA1) 10 (AYA2) 6.3

8

7

9.5

9

pH

Activity

94 kDa

96.32 kDa

121 kDa

25 kDa (AYA-1) 31 kDa (AYA-2)

115 kDa

~90 kDa

35 kDa

–

Molecular weight

37

45

37

30

37

37

47 (AYA1) 67 (AYA2) 30

50

30

50

30

Temp (°C)

M

Elshafei et al. (2012)

–

2.56 μM

3380.0 μmol/ min.mg 279.27 U/mL

–

4.00 × 10−3 M 0.0253 mg/mL

–

Lincoln et al. (2015)

Shrivastava et al. (2012) Patro and Gupta (2012) Huang et al. (2014)

Kumar and Manonmani (2013) Thakur et al. (2014)

4.44 μmol/ mL.min 625 U/mL

Ahmed et al. (2015)

Dias et al. (2016)

Vala et al. (2018)

Dange and Penshwe (2011) Dutta et al. (2015)

Reference

–

6.228 μmol/ mg. min 313 IU/mL

355.3 μmol/ mg. min

104.16 IU/ mL

Vmax

1 × 10–5 M

1.05 mM

4.3 mM

0.1 M

–

0.66 × 10−3 M

0.8141 mM

7.02 × 10

−3

12.5 × 10−3 M

Km

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or molecular exclusion with Sephadex (Tundisi et al., 2016). The Lasparaginase from Penicillium digitatum was purified about 60.95 fold after precipitation with ammonium sulfate 70–80% saturation and Sephadex G-100 column chromatography obtaining 833.15 IU/mg of specific activity and 4.35% of recovery (Shrivastava et al., 2012) whereas Ahmed, Dahab, M, and SM, (2015) purified two types of Lasparaginase (AYA-1 and AYA-2) from the culture supernatant of Aspergillus sp. ALAA-2000 through ammonium sulfate precipitation and Sephadex G-200 columm, obtaining purification factor of 8.3 fold. Affinity chromatography using a column of nickel-iminiodiacetic acid has also been used for the purification of fungal L-asparaginase and the purification yield obtained was 2.6 protein specific activity of 1984.8 IU/mg (Huang, Liu, Sun, Yan, & Jiang, 2014). In Table 3, different methods of purification and biochemical characterization for various fungal L-asparaginases are reported according to the literature. L-asparaginases from different microorganisms vary in their biochemical properties. Generally, the optimum temperature for L-asparaginase activity is between 30 and 50 °C (Huang et al., 2014; Kumar & Manonmani, 2013; Lincoln et al., 2015). In relation to the optimum pH, L-asparaginase presents activity in a wide range of pH, with optimum activity in the range of 6.0 to 9.5 (Dias et al., 2016; Dutta et al., 2015; Vala et al., 2018). However, most fungal L-asparaginases show optimum activity at alkaline pH, while only a few strains have optimum pH below pH 7.0 (Ahmed, Dahab, M, & SM, 2015; Kumar & Manonmani, 2013). Purified L-asparaginase from Aspergillus aculeatus showed maximum activity at pH 9.0 and 30 °C. The enzyme retained more then 70% activity after 10 min at 50 °C while it was inactivated at 90 °C (Dange & Penshwe, 2011). Purified L-asparaginase from Aspergillus oryzae CCT 3940 showed high stability under physiological conditions, remaining stable in the pH range of 7.0 to 8.0 after 1 h incubation in the temperature range of 30 to 45 °C (Dias et al., 2016). Ions, as well as activating and inhibiting substances, can affect the activity of L-asparaginase from different fungal sources. L-asparaginase from Mucor hiemalis was activated 100% by Mn2+ at 2 mM concentration, after incubation at 37 °C for 30 min and around 100 to 150% activation in the presence of the ions Ba2+, K+, Cu2+, Mn2+, and Hg2+. The enzyme was inhibited by around 50 to 100% in the presence of Fe2+ and Na+. However, ethylenediamine tetraacetic acid (EDTA) at 2 mM concentration did not affect the enzymatic activity, suggesting that the enzyme is not metalloenzyme. Tween 80 and Triton X-100 surfactants (2 mM) activated about 350% of the residual activity of Lasparaginase while sodium docetil sulfate (SDS) (2 mM) inhibited the enzyme, obtaining 17% of the residual activity (Thakur et al., 2014). Vala et al. (2018) found that 1% Tween 80 and Triton X-100 increased around 100 and 120% of L-asparaginase activity from Aspergillus niger AKV-MKBU, respectively. The enzyme was inhibited by As+, Cr6+, Mn2+, Hg2+, and Sn2+ at the concentration of 10 mM. L-asparaginase was completely inhibited by 2-mercaptoethanol (2 mM) and heavily inhibited by urea (2 mM), thiourea (2 mM), SDS (2%), and EDTA (2 mM). Lincoln et al. (2015) analyzed the effect of different compounds on Trichoderma viride L-asparaginase activity. The enzyme was tested with metal ions, inhibitors, and activators at 5 mM concentration and incubated for 30 min at 37 °C. L-asparaginase was activated at about 140 and 160% by Mg2+ and Na+ and inhibited by Fe2+, Fe3+, Co2+, and Mn2+. The enzyme was strongly inhibited by EDTA. Nethylemaleimide and phenylmethylsulphonylfluoride did not alter the enzyme activity.

optimization of experiments design and artificial neural network coupled with a genetic algorithm to find the significant components of the fermentation and optimum concentration for L-asparaginase production by Aspergillus terreus MTCC 1782 by submerged fermentation. The optimization of the components of the fermentation medium using genetic algorithm connected to an artificial neural network was shown to be more effective than the regression model of the response surface methodology. The optimum predicted concentration of the components of the medium using the artificial neural network was 1.7% of L-proline, 1.99% of sodium nitrate, 1.38% of L-asparagine, and 0.65% of glucose with the experimental production of 40.85 IU/mL of L-asparaginase. In another study, Dorya and Kumar (2018) used a sequential strategy to optimize L-asparaginase production using Aspergillus sp. In the first stage, through blending planning, maximum L-asparaginase activity was observed using a ternary mixture of cotton seed cake (2/3), wheat bran (1/6) and red bean skin (1/6). In the next step, the culture parameters were optimized using the Box-Behnken design. After 6 days of fermentation using optimized ternary mixture, the maximum activity of 12.57 U/mL L-asparaginase was obtained at 35 °C, pH 8, and humidity of 70% (w/v). The authors observed that through the sequential optimization study the production of L-asparaginase increased 1.3 fold. Dias et al. (2015) used a Simplex Mixture design to investigate the presence of synergistic or antagonistic effects of different agroindustrial residues to produce L-asparaginase under solid-state fermentation by Aspergillus niger LBA 02. The researchers obtained the highest L- asparaginase activity (89.22 U/g) after 96 h of fermentation using a ternary mixture composed of wheat bran (1/3), soybean meal (1/3), and cottonseed (1/3). This process showed maximization of L-asparaginase production when the blends were used in comparison with those in isolation. Uppuluri et al. (2013) optimized the conditions to produce L-asparaginase by Aspergillus niger C4 in a bioreactor using black sesame oil bagasse as a substrate for solid-state fermentation. The authors used the Box-Behnken design for three variables (aeration, bed thickness, and temperature). Statistical analysis revealed a maximal L-asparaginase yield of 310 U/gds. Where the aeration parameters, bed thickness, and temperature were 0.44 vvm, 17.35 cm and 29.31 °C, respectively. Baskar et al. (2010) applied the Latin Square design to find the best nitrogen source for the extracellular production of L-asparaginase by Aspergillus terreus MTCC 1782 using corn flour as substrate in submerged fermentation. Urea was identified as the best nitrogen source with a mean L-asparaginase production of 33.25 IU/mL. 2.3. Purification and biochemical properties of fungal L-asparaginase Most purification procedures are performed using conventional methods, such as ammonium sulfate fractionationing combined with molecular exclusion chromatography or ion exchange column chromatography (Zuo et al., 2015). Most studies use (NH4)2SO4 to precipitate the L-asparaginase (Dange & Penshwe, 2011; Dias, Ruiz, Torre, & Sato, 2016; Dutta, Ghosh, & Pramanik, 2015; Mishra, 2006). The salt concentration ranges from 35% to 100% depending on the L-asparaginase source (Lopes et al., 2017). The use of organic solvents or short chain alcohols, such as methanol and ethanol, are also used for the precipitation of L-asparaginase (Tundisi et al., 2016). Other precipitating agents, such as acetone, are also used (Lincoln et al., 2015; Thakur et al., 2014). Chromatographic methods are frequently used to achieve maximum purification. The ion exchange chromatography with DEAE cellulose is the most popular chromatographic method (Tundisi et al., 2016). Lincoln et al. (2015) when purifying Trichoderma viride L-asparaginase using DEAE-cellulose had purification fold of 13, while Vala et al. (2018) purified L-asparaginase obtained from Aspergillus niger AKVMKBU and obtained 10.36 of purification fold. Another commonly used technique is gel filtration chromatography

2.4. Formation of acrylamide and application of fungal L-asparaginase in food industry In 2002, the Swedish National Food Administration (SNFA) reported the presence of acrylamide in food with high carbohydrate content subjected to elevated temperatures (Mottram, Wedzicha, & Dodson, 2002; Tareke, Rydberg, Karlsson, Eriksson, & Törnqvist, 2002). 6

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its precursors or to inhibit or reduce the intensity of the Maillard reaction by different process modifications (Pedreschi, 2009). Most of the methods used to reduce this toxic compound have a negative impact on both taste and appearance of the final product (Batool et al., 2016). The use of fungic L-asparaginase presents a relatively new, promising, and great technique to reduce the acrylamide levels in food. This enzyme helps with mitigation strategies from two aspects, interference with the Maillard reaction or the removal of precursors by converting L-asparagine into L-aspartic acid (non-toxic) without altering the nutritional value, appearance, or flavor of the final product (Batool et al., 2016; Hendriksen et al., 2009; Swanston, 2018). L-asparaginase may be used as a food additive to achieve a technological purpose during manufacture. The enzyme should be removed from the food or inactivated, however, the presence of traces of the substance or its derivatives is permitted. Residues of the enzyme, including denatured L-asparaginase, may range from 0.14 to 428 mg/kg of food (JECFA, 2009).

Since then, research institutions and food agencies have been investigating the toxicity, formation, mitigation, and detection of acrylamide in food (Hu, Xu, Fu, & Li, 2015). Food related to human exposure to acrylamide are potato, coffee, and bakery products (biscuits and bread) (EFSA, 2015). The estimate of dietary intake was studied in several populations with different eating habits (Dybing et al., 2005; Hilbig, Freidank, Kersting, Wilhelm, & Wittsiepe, 2004; Hilbig & Kersting, 2006). The intake of acrylamide in the diet is estimated to be between 0.3 and 1.9 μg/kg orally (EFSA, 2015). However, food with the highest contribution of acrylamide intake varies from country to country, according to dietary patterns and preparation methods (Claus, Carle, & Schieber, 2008). The main mechanism that has been accepted by researchers to explain the formation of acrylamide in food involves the Maillard reaction, from the reaction between the food components as amino acids and reducing sugars under heat treatment (Cladiere & Camel, 2017; Mottram et al., 2002; Pedreschi, 2009; Yaylayan & Stadler, 2005). The first step in this reaction is the intermediate formation of the Schiff base. Subsequently, this Schiff base can be hydrolyzed to form 3-aminopropionamide, precursor of acrylamide or undergo elimination of an amide grouping to directly form acrylamide (Claus et al., 2008), according to Fig. 3. Several observations have led to the hypothesis that food heating could be an important source of human exposure to acrylamide. Heating processes such as frying and baking promote the formation of acrylamide, whereas this compound was not detected in cooked food (Keramat, LeBail, Prost, & Jafari, 2011). The factors that influence the formation of acrylamide in food are: processing conditions (temperature, humidity, cooking time, and product matrix) and precursors such as reducing sugars and free amino acids (mainly L-asparagine) (Zuo et al., 2015). However, most of the methods used to attenuate the formation of acrylamide seek to remove

2.4.1. French fries Products derived from potatoes (Solanum tuberosum), French fries are widely consumed in many countries. Fried potato products have contributed to 50% of the human ingestion of acrylamide in European countries (Keramat et al., 2011; Mesias, Delgado-Andrade, Holgado, & Morales, 2018). The amino acid L-asparagine is recognized as the main precursor for the formation of acrylamide in products derived from potatoes, so it is important to verify the performance of the enzyme in the decrease of this compound, since a high content of this amino acid in potatoes may be also related to the increase of the acrylamide formation (Becalski et al., 2004; Becalski, Lau, Lewis, & Seaman, 2003). According to a study by Mesias et al. (2018), the potential of acrylamide formation in potatoes is related to several factors such as: Lasparagine content and reducing sugars, color, and moisture (in fresh

Fig. 3. Mechanism of acrylamide formation in heated foods. 7

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acrylamide in biscuits by up to 70% using commercial L-asparaginase. These authors also did not observe alteration of color or flavor. Anese, Quarta, Peloux, and Calligaris (2011) studied the influence of biscuit composition on the ability of L-asparaginase (Novozymes®) to minimize the formation of acrylamide. Different formulations of biscuits were tested, varying the water composition and fat type and adding 900 U/Kg of flour in all treatments. The authors concluded that differences in the efficiency of L-asparaginase, may be related to the different compositions of the evaluated food. Despite being able to remove acrylamide, L-asparaginase showed a better reduction of acrylamide in biscuits with high water content, however, the reduction of acrylamide decreased with increasing fat content in biscuits. In another study, the effect of L-asparaginase on the formation of acrylamide in biscuits using experimental modeling models was evaluated. Anese, Quarta, and Frias (2011) found that the use of intermediate concentrations of L-asparaginase (500 U/Kg), the formation of acrylamide was minimal, as well as in the shortest time and the lowest incubation temperature. The important parameter of the cookie, color, was not affected by the application of the enzyme. The L-asparaginase gene from Rhizomucor miehei was cloned and expressed in Escherichia coli by Huang et al. (2014), the enzyme (10 U/ mg flour) was used to reduce the level of acrylamide in biscuits, reaching approximately 80% reduction. In addition, the enzyme was useful in reducing the acrylamide present in breads and also demonstrated potential for use in the treatment of leukemia. There is a strong correlation between color intensity of the crust and formation of acrylamide, particularly when bread is baked at temperatures above 200 °C (Keramat et al., 2011). Mohan Kumar, Shimray, Indrani, and Manonmani (2014) applied L-asparaginase produced from Cladosporium sp. in sweet bread at different concentrations (50–300 U). The authors did not observe changes in the physical-sensorial characteristics of the bread with treatment with L-asparaginase. In the treatment with 300 U, there were 97% and 73% reduction of the formation of acrylamide in the crust and crumb bread regions, respectively. These results indicated the potential of L-asparaginase for industrial and domestic applications for acrylamide reduction.

potatoes), oil and utensils used for frying, content of polar compounds in the frying oil (during the frying process), color, thickness, and visual color (after frying). Other relevant factors related to the formation of acrylamide in potatoes are: potato variety, soil type, fertilization, weather, storage, cutting, blanching, and drying process, and use of additives (Muttucumaru et al., 2017; Vinci, Mestdagh, & de Meulenaer, 2012). Though all those factors are related to acrylamide formation, reducing sugars and L-asparagine are the limiting factor for acrylamide formation in potato products (Muttucumaru et al., 2017; Williams, 2005). Acrylamide reduction in potato chips was tested under different conditions using, in addition to the treatments alone, combinations of bleaching and L-asparaginase from Aspergillus oryzae. The largest reduction (90%) was achieved when bleaching (85 °C/3.5 min) was applied followed by immersion in L-asparaginase solution (50 °C/20 min). The heat treatment was able to cause changes in the structure of the potatoes, facilitating the diffusion of the enzyme into the tissues and, consequently, favoring and enhancing its action (Pedreschi, Mariotti, Granby, & Risum, 2011). Another study that reported the use of the fungi L-asparaginase from Aspergillus oryzae CCT 3940 (50 U/mL) to mitigate acrylamide from French fries showed a 72% acrylamide reduction (after a frying process at 180 °C for 7 min) compared to a control sample (Dias, Bogusz, Hantao, Augusto, & Sato, 2017). An L-asparaginase produced by Fusarium culmorum (ASP-87) was used to reduce acrylamide in potato products. The potato chips was treated with the enzyme at 40 °C for 30 min, followed by frying at temperatures of 170–180 °C for 90 s. It was observed that 300 U/L of Lasparaginase was required to reduce 85% and 94% of acrylamide levels in potato chips and French fries, respectively. L-asparaginase was efficient in inhibiting the formation of acrylamide as well as L-asparagine decrease. The authors also found high levels of acrylamide in different types of products obtained in the local market (Bengaluru, India). Values higher than 3020 μg/kg were observed for potato chips and 4475 μg/kg for French fries (Meghavarnam & Janakiraman, 2018). 2.4.2. Bakery products The color of bakery products is one of the most important attributes that, in addition to affecting food quality, influences consumer acceptance (Bartkiene et al., 2016; Lu & Zheng, 2012). Maillard's reaction is involved in the formation of the specific sensory attributes of these products, and acrylamide is also formed during the reaction (Thomas & Thomas, 2014). In Europe, 20% of the intake of acrylamide has been attributed to bakery products (Keramat et al., 2011). In 2013, the Brazilian Association of Consumer Protection (Proteste, 2013) evaluated the amount of acrylamide in 51 products from eight categories of food - potato chips, sweet and savory biscuits, cream crackers, French bread, snacks and toast - and found that French bread and sweet and biscuits presented the highest values in acrylamide content. Sweet biscuits, for example, presented values between 1100 mg/kg and < 100 mg/kg acrylamide. According to Nguyen, Fels-Klerx, Peters, and Boekel (2016), free Lasparagine in wheat flour was the main factor responsible for the formation of acrylamide in biscuits with reduced sugar content, and free Lasparagine can be a limiting factor for the formation of acrylamide in cereal products. Some studies have been carried out on the application of fungal Lasparaginase to reduce acrylamide in starch products. Hendriksen et al. (2009) applied Aspergillus oryzae L-asparaginase in sweet and savory biscuits, the authors obtained a 34–92% reduction in the acrylamide levels of these products. A similar result was obtained by Ciesarova, Kukurová, Bednáriková, Marková, and Baxa (2009) who obtained a reduction of > 97% in the acrylamide content, by applying the commercial L-asparaginase enzyme to gingerbread without damaging the sensorial quality of the final product. Vass, Amrein, Schönbächler, Escher, and Amado (2004) have been able to reduce the contents of

2.4.3. Coffee Together with potatoes and cereal products, roasted coffee is one of the products that have high concentration of acrylamide (Mesias et al., 2018). The two main coffee species used in the preparation of the beverage are arabica (Coffea arabica) and robusta (Coffea canefora robusta). The quality of the drink depends on the proportion of each coffee bean (Alves, Soares, Casal, Fernandes, & Oliveira, 2010). High concentrations of acrylamide are found in roasted robusta coffee beans in relation to roasted arabica coffee beans (Anese, 2016). According to the results of the EFSA Contaminants Panel in the Food Chain (CONTAM) between 2010 and 2013 > 1500 coffee-based products were analyzed, and the average acrylamide concentration found was 578 ng/g in roasted coffee (EFSA, 2015). Coffee has high levels of acrylamide due to the roasting process. Storage conditions may also contribute to the formation of acrylamide (Mesias & Morales, 2016). Exposure to acrylamide, from roasted coffee consumption, varies from country to country, age, and sex of the consumer, roast grade and volume of coffee ingested, and so on (Anese, 2016). Consequently, daily intake of this product represents a significant source of exposure to acrylamide (Şenyuva & Gökmen, 2005). Another problem is related to the fact that acrylamide is a polar molecule. The preparation of ground coffee with hot water may allow the complete extraction of the acrylamide present in the roasted coffee beans (Guenther, Anklam, Wenzl, & Stadler, 2007). One of the preventive measures of acrylamide formation is the use of L-asparaginase to maintain the lowest concentration of this compound during roasting, acting on precursors such as L-asparagine, creating less favorable reaction conditions (Anese, 2016). In the study by Hendriksen, Budolfsen, and Baumann (2013), the effect of L8

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asparaginase on reduction of acrylamide in coffee was evaluated. Green arabica beans were steamed with water for 45 min at 100 °C and treated with L-asparaginase at 60 °C for 60 min. The authors observed the greatest reduction when they used the concentration of 6000 ASU/kg of grains, observing a reduction of 70–80% in the content of L-asparagine and 55–74% in the formation of acrylamide.

acrylamide. Environnement, Risques & Santé, 16, 31–43. Claus, A., Carle, R., & Schieber, A. (2008). Acrylamide in cereal products: A review. Journal of Cereal Science, 47, 118–133. Cunha, M. C., Silva, L. C., Sato, H. H., & Castro, R. J. S. (2018). Using response surface methodology to improve the L-asparaginase production by Aspergillus niger under solid-state fermentation. Biocatalysis and Agricultural Biotechnology, 16, 31–36. Dange, V. U., & Penshwe, S. A. (2011). Production, purification and characterization of fungal L-asparaginase. Boiano Frontier, 4, 162–167. Dias, F. F. G., Bogusz, S., Hantao, L., Augusto, F., & Sato, H. H. (2017). Acrylamide mitigation in French fries using native L-asparaginase from Aspergillus oryzae CCT 3940. LWT - Food Science and Technology, 76, 222–229. Dias, F. F. G., Castro, R. J. S., Ohara, A., Nishide, T. G., Bagagli, M. P., & Sato, H. H. (2015). Simplex centroid mixture design to improve L-asparaginase production in solid-state fermentation using agroindustrial wastes. Biocatalysis and Agricultural Biotechnology, 4, 528–534. Dias, F. F. G., Ruiz, A. L. T., Torre, A. D., & Sato, H. H. (2016). Purification, characterization and antiproliferative activity of L-asparaginase from Aspergillus oryzae CCT 3940 with no glutaminase activity. Asian Pacific Journal of Tropical Biomedicine, 6, 785–794. Dias, F. F. G., & Sato, H. H. (2016). Sequential optimization strategy for maximum Lasparaginase production from Aspergillus oryzae CCT 3940. Biocatalysis and Agricultural Biotechnology, 6, 33–39. Doriya, K., & Kumar, D. S. (2016). Isolation and screening of L-asparaginase free of glutaminase and urease from fungal sp. 3 Biotech, 6, 229–239. Dorya, K., & Kumar, D. S. (2018). Optimization of solid substrate mixture and process parameters for the production of L-asparaginase and scale-up using tray bioreactor. Biocatalysis and Agricultural Biotechnology, 13, 244–250. DSM (2018). DSM introduces PreventASe® XR to extend use of enzymatic acrylamidereduction solution to more snacks and baked goods. Retrieved 2019-01-24. Home Page https://www.dsm.com/corporate/media/informationcenter-news/2018/07/ 2018-07-10-dsm-introduces-preventase-xr-to-extend-use-of-enzymatic-acrylamidereduction-solution-to-more-snacks-and-baked-goods1.html. Dutta, S., Ghosh, S., & Pramanik, S. (2015). L-asparaginase and L-glutaminase from Aspergillus fumigatus WL002: Production and some physicochemical properties. Applied Biochemistry and Microbiology, 51, 425–431. Dybing, E., Farmer, P. B., Andersen, M., Fennell, T. R., Lalljie, S. P. D., ... Verger, P. (2005). Human exposure and internal dose assessments of acrylamide in food. Food and Chemical Toxicology, 43, 365–410. El-Naggar, N. E. A., El-Ewasy, S., & El-Shweihy, N. M. (2014). Microbial L-asparaginase as a potential therapeutic agent for the treatment of acute lymphoblastic leukemia: The pros and cons. International Journal of Pharmaceutics, 10, 182–199. Elshafei, A. M., & El-Ghonemy, D. H. (2015). Screening and media optimization for enhancing L-asparaginase production, an anticancer agent, from different filamentous fungi in solid-state fermentation. British Biotechnology Journal, 9, 1–15. Elshafei, A. M., Hassan, M. M., Abouzeid, M. A. E., Mahmoud, D. A., & Elghonemy, D. H. (2012). Purification, characterization and antitumor activity of L-asparaginase from Penicillium brevicompactum NRC 829. British Microbiology Research Journal, 2, 158–174. European Food Safety Authority. EFSA CONTAM Panel (EFSA Panel on Contaminants in the Food Chain) (2015). Scientific opinion on acrylamide in food. EFSA Journal, 2015. Retrieved 2017-01-26. Home Page http://www.efsa.europa.eu/efsajournal. Farag, A. M., Hassan, S. W., Beltagy, E. A., & El-Shenawy, M. A. (2015). Optimization of production of anti-tumor L-asparaginase by free and immobilized marine Aspergillus terreus. Egyptian Journal of Aquatic Research, 41, 295–302. Friedman, M. (2015). Acrylamide: Inhibition of formation in processed food and mitigation of toxicity in cells, animals, and humans. Food & Function, 6, 1752–1772. Guenther, H., Anklam, E., Wenzl, T., & Stadler, R. H. (2007). Acrylamide in coffee: Review of progress in analysis, formation and level reduction. Food Additives and Contaminants, 24, 60–70. Heinemann, B., & Howard, A. J. (1969). Production of tumor-inhibitory L-asparaginase by submerged growth of Serratia marcescens. Applied Microbiology, 18, 550–554. Hendriksen, H. V., Budolfsen, G., & Baumann, M. J. (2013). Asparaginase for acrylamide mitigation in food. Aspects of Applied Biology, 116, 41–50. Hendriksen, H. V., Kornbrust, B. A., Østergaard, P. R., & Stringer, M. A. (2009). Evaluating the potential for enzymatic acrylamide mitigation in a range of food products using an asparaginase from Aspergillus oryzae. Journal of Agricultural and Food Chemistry, 57, 4168–4176. Hilbig, A., Freidank, N., Kersting, M., Wilhelm, M., & Wittsiepe, J. (2004). Estimation of the dietary intake of acrylamide by German infants, children and adolescents as calculated from dietary records and available data on acrylamide levels in food groups. International Journal of Hygiene and Environmental Health, 207, 463–471, 2004. Hilbig, A., & Kersting, M. (2006). Dietary acrylamide exposure, time trends and the intake of relevant foods in children and adolescents between 1998 and 2004: Results of the DONALD study. Journal für Verbraucherschutz und Lebensmittelsicherheit, 1, 10–18, 2006. Hu, Q., Xu, X., Fu, Y., & Li, Y. (2015). Rapid methods for detecting acrylamide in thermally processed foods: A review. Food Control, 56, 135–146. Huang, L., Liu, Y., Sun, Y., Yan, Q., & Jiang, Z. (2014). Biochemical characterization of a novel L-asparaginase with low glutaminase activity from Rhizomucor miehei and its application in food safety and leukemia treatment. Applied and Environmental Microbiology, 80, 1561–1569. Hymavathi, M., Sathist, T., Rao, C. S., & Prakasham, R. S. (2009). Enhancement of Lasparaginase production by isolated Bacillus circulans (MTCC 8574) using response surface methodology. Applied Biochemistry and Biotecnology, 159, 191–198. IARC (International Agency for Research on Cancer) (1994). Acrylamide. IARC

3. Conclusion L-asparaginase is of great importance in food applications. Research aimed at the discovery of new L-asparaginases, mainly those produced by fungi, associated with the use of statistical methodologies of optimization and the use of less laborious fermentative processes, have great potential to obtain cheaper enzymes with desirable properties for application in food aiming at the reduction of acrylamide and in the future, for the treatment of some types of cancer. Acknowledgments This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) Finance Code 001 (PROEX process number 23038.000795/2018-61). The authors would like to the Department of Food Science, School of Food Engineering, University of Campinas, which are gratefully acknowledged. References Ahmed, M. M. A., Dahab, N. A., M, T. T., & SM, F. H. (2015). Production, purification and characterization of L-asparaginase from marine endophytic Aspergillus sp. ALAA-2000 under submerged and solid state fermentation. Journal of Microbial and Biochemical Technology, 7, 165–172. Alves, R. C., Soares, C., Casal, S., Fernandes, J. O., & Oliveira, M. B. P. P. (2010). Acrylamide in espresso coffee: Influence of species, roast degree and brew length. Food Chemistry, 119, 929–934. Anese, M. (2016). Acrylamide in coffee and coffee substitutes. Acrylamide in Food, 181–195. Anese, M., Quarta, B., & Frias, J. (2011). Modelling the effect of asparaginase in reducing acrylamide formation in biscuits. Food Chemistry, 126, 435–440. Anese, M., Quarta, B., Peloux, L., & Calligaris, S. (2011). Effect of formulation on the capacity of L-asparaginase to minimize acrylamide formation in short dough biscuits. Food Research International, 44, 2837–2842. Bartkiene, E., Jakobsone, I., Pugajeva, I., Bartkevics, V., Zadeike, D., & Juodeikiene, G. (2016). Reducing of acrylamide formation in wheat biscuits supplemented with flaxseed and lupine. LWT - Food Science and Technology, 65, 275–282. Baskar, G., & Renganathan, S. (2010). Optimization of media components and operating conditions for exogenous production of fungal L-asparaginase. Chiang Mai Journal of Science, 38, 270–279. Baskar, G., & Renganathan, S. (2011). Statistical and evolutionary optimization of operating conditions for enhanced production of fungal L-asparaginase. Chemical Papers, 65, 798–804. Baskar, G., & Renganathan, S. (2012). Optimization of L-asparaginase production by Aspergillus terreus MTCC 1782 using response surface methodology and artificial neural network-linked genetic algorithm. Asia-Pacific Journal of Chemical Engineering, 7, 212–220. Baskar, G., Sriharini, C., Sripriya, R., & Renganathan, S. (2010). Statistical screening of supplementary nitrogen source for enhanced production of L-asparaginase by Aspergillus terreus 1782. Chemical and Biochemical Engineering Quarterly, 24, 467–472. Batool, T., Makky, E., Jalal, M., & Yusoff, M. (2016). A comprehensive review on Lasparaginase and its applications. Applied Biochemistry and Biotecnology, 178, 900–923. Becalski, A., Lau, B. P. Y., Lewis, D., & Seaman, S. W. (2003). Acrylamide in foods: Occurrence, sources, and modeling. Journal of Agricultural and Food Chemistry, 51, 802–808. Becalski, A., Lau, B. P. Y., Lewis, D., Seaman, S. W., Hayward, S., ... Leclerc, Y. (2004). Acrylamide in French fries: Influence of free amino acids and sugars. Journal of Agricultural and Food Chemistry, 52, 3801–3806. Cachumba, J. J. M., Antunes, F. A. F., Peres, G. F. D., Brumano, L. P., Santos, J. C., & Silva, S. S. (2016). Current applications and different approaches for microbial L-asparaginase production. Brazilian Journal of Microbiology, 47, 77–85. Capuano, E., & Fogliano, V. (2011). Acrylamide and 5-hydroxymethylfurfural (HMF): A review on metabolism, toxicity, occurrence in food and mitigation strategies. LWT Food Science and Technology, 44, 793–810. Ciesarova, Z., Kukurová, K., Bednáriková, A., Marková, L., & Baxa, L. (2009). Improvement of cereal product safety by enzymatic way of acrylamide mitigation. Czech Journal of Food Sciences, 27, 96–98. Cladiere, M., & Camel, V. (2017). The Maillard reaction and food safety: Focus on

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M.C. da Cunha, et al.

Rivas (Vol. Ed.), Processing effects on safety and quality of foods. Vol. 310. Processing effects on safety and quality of foods (pp. 231–251). USA: Taylor and Francis. Pedreschi, F., Kaack, K., & Granby, K. (2008). The effect of asparaginase on acrylamide formation in French fries. Food Chemistry, 109(2), 386–392. Pedreschi, F., Mariotti, S., Granby, K., & Risum, J. (2011). Acrylamide reduction in potato chips by using commercial asparaginase in combination with conventional blanching. LWT - Food Science and Technology, 44(6), 1473–1476. Pennisi, M., Malaguarnera, G., Puglisi, V., Vinciguerra, L., Vacante, M., & Malaguarnera, M. (2013). Neurotoxicity of acrylamide in exposed workers. International Journal of Environmental Research and Public Health, 10, 3843–3854. Proteste (2013). Acrilamida: consuma a menor quantidade possível. Retrieved 2017-0302. Home Page https://www.proteste.org.br/nt/nc/artigo/acrilamida-27. Sarquis, M. I. M., Oliveira, E. M. M., Santos, A. S., & Costa, G. L. (2004). Production of Lasparaginase by filamentous fungi. Memories of the Oswaldo Cruz Institute, 99, 489–492. Şenyuva, H. Z., & Gökmen, V. (2005). Study of acrylamide in coffee using an improved liquid chromatography mass spectrometry method: Investigation of colour changes and acrylamide formation in coffee during roasting. Food Additives and Contaminants, 22(3), 214–220. Shrivastava, A., Khan, A. A., Shrivastav, A., Jain, S. K., & Singhal, P. K. (2012). Kinectis studies of L-asparaginase from Penicillium digitatum. Preparative Biochemistry and Biotechnology, 42, 574–581. Singhania, R. R., Patel, A. K., Soccol, C. R., & Pandey, A. (2009). Recent advances in solidstate fermentation. Biochemical Engineering Journal, 44, 13–18. Souza, P. M., Freitas, M. M., Cardoso, S. L., Pessoa, A., Guerra, E. N. S., & Magalhães, P. O. (2017). Optimization and purification of L-asparaginase from fungi: A systematic review. Critical Reviews in Oncology/Hematology, 120, 194–202. Stanbury, P. F., Whitaker, A., & Hall, S. J. (1995). Principles of fermentation technology (2nd ed.). Oxford: Butterworth Heinemann. Swanston, J. (2018). Acrylamide mitigation in foods—an update. Retrieved 2018-06-07. Home Page: https://www.leatherheadfood.com/files/2018/01/White-paper-59Acrylamide-in-foods-an-update.pdf. Tareke, E., Rydberg, P., Karlsson, P., Eriksson, S., & Törnqvist, M. (2002). Analysis of acrylamide, a carcinogen formed in heated foodstuffs. Journal of Agricultural and Food Chemistry, 50, 4998–5006. Thakur, M., Lincoln, L., Niyonzima, F. N., & More, S. S. (2014). Isolation, purification and characterization of fungal extracelular L-asparaginase from Mucor hiemalis. Journal of Biocatalysis & Biotransformation, 2, 1–9. Thomas, A., & Thomas, A. (2014). Acrylamide—a potent carcinogen in food. International Journal of Science and Research, 3, 177–180. Thomas, L., Larroche, C., & Pandey, A. (2013). Current developments in solid-state fermentation. Biochemical Engineering Journal, 81, 146–161. Tundisi, L. L., Coêlho, D. F., Zanchetta, B., Moriel, P., Pessoa, A., Jr., ... Silveira, E. (2016). L-asparaginase purification. Separation and Purification Reviews, 46, 35–43. Uppuluri, K. B., Dasari, R. K. V. R., Sajja, V., Jacob, A. S., & Reddy, D. S. R. (2013). Optimization of L-asparaginase production by isolated Aspergillus niger C4 from sesame (black) oil cake under SSF using Box-Behnken design in column bioreactor. International Journal of Chemical Reactor Engineering, 11, 103–109. Vala, A. K., Sachaniya, B., Dudhagara, D., Panseriya, H. Z., Gosai, H., ... Dave, B. P. (2018). Characterization of L-asparaginase from marine derived Aspergillus niger AKVMKBU, its antiproliferative activity and bench scale production using industrial waste. International Journal of Biological Macromolecules, 107, 41–46. Vass, M., Amrein, T. M., Schönbächler, B., Escher, F., & Amado, R. (2004). Ways to reduce acrylamide formation in cracker products. Czech Journal of Food Science, 22, 19–21. Vinci, R. M., Mestdagh, F., & de Meulenaer, B. (2012). Acrylamide formation in fried potato products—present and future, a critical review on mitigation strategies. Food Chemistry, 133(4), 1138–1154. Williams, J. S. E. (2005). Influence of variety and processing conditions on acrylamide levels in fried potato crisps. Food Chemistry, 90(4), 875–881. Xu, F., Oruna-Concha, M.-J., & Elmore, J. S. (2016). The use of asparaginase to reduce acrylamide levels in cooked food. Food Chemistry, 210, 163–171. Yaylayan, V. A., & Stadler, R. H. (2005). Acrylamide formation in food: A mechanistic perspective. Journal of AOAC International, 88, 262–267. Zuo, S., Zhang, T., Jiang, B., & Mu, W. (2015). Recent research progress on microbial Lasparaginases. Applied Microbiology and Biotechnology, 99, 1069–1079.

monographs on the evaluation of carcogenic risks to humans. Vol. 60. Izadpanah, F., Homaei, A., Fernandes, P., & Javadpour, S. (2018). Marine microbial Lasparaginase: Biochemistry, molecular approaches and applications in tumor therapy and in food industry. Microbiological Research, 208, 99–112. JECFA (2007). Compendium of food additive specifications. Monograph N°. 4. Rome: FAO. JECFA (2009). Evaluation of certain food additives: Sixty-ninth report of the joint FAO/WHO expert committee on food additives. WHO Technical Report SeriesFAO: Rome. Jha, S. K., Pasrija, D., Sinha, R. K., Singh, H. S., Nigan, V. K., & Vidyarthi, A. S. (2012). Microbial L-asparaginase: A review on current scenario and future prospects. International Journal of Phamaceutical Sciences and Research, 3, 3076–3090. Kaliwal, B. B., & Hosamani, R. (2011). Isolation, molecular identification and optimization of fermentation parameters for the production of L-asparaginase, an anticancer agent by Fusarium equiset. International Journal of Microbiology Research, 3, 108–119. Keramat, J., LeBail, A., Prost, C., & Jafari, M. (2011). Acrylamide in baking products: A review article. Food and Bioprocess Technology, 4, 530–543. Kumar, N. S., & Manonmani, H. K. (2013). Purification, characterization and kinetic properties of extracellular L-asparaginase produced by Cladosporium sp. World Journal of Microbiology and Biotecnhology, 29, 577–587. Lincoln, L., Niyonzima, F. N., & More, S. S. (2015). Purification and properties of a fungal L-asparaginase from Trichoderma viride pers: sf grey. Journal of Microbiology, Biotechnology and Food Sciences, 4, 310–316. Lopes, A. M., Oliveira-Nascimento, L., Ribeiro, A., Tairum, C. A., Jr., Breyer, A. C., ... Pessoa, A. (2017). Therapeutic L-asparaginase: Upstream, downstream and beyond. Critical Reviews in Biotechnology, 37, 82–99. Lu, H., & Zheng, H. (2012). Fractal colour: A new approach for evaluation of acrylamide contents in biscuits. Food Chemistry, 134, 2521–2525. Mashburn, L. T., & Wriston, J. C., Jr. (1964). Tumor inhibitory effect of L-asparaginase from Escherichia coli. Archives of Biochemistry and Biophysics, 105, 450–453, 1964. Maziero, M., & Bersot, L. (2010). Mycotoxins in food produced in Brazil. Brazilian Journal of Agroindustrial Products, 12, 89–99. Meghavarnam, A. K., & Janakiraman, S. (2017). Solid-state fermentation: An effective fermentation strategy for the production of L-asparaginase by Fusarium culmorum (ASP-87). Biocatalysis and Agricultural Biotechnology, 11, 124–130. Meghavarnam, A. K., & Janakiraman, S. (2018). Evaluation of acrylamide reduction potential of L-asparaginase from Fusarium culmorum (ASP-87) in starchy products. LWT - Food Science and Technology, 89, 32–37. Mesias, M., Delgado-Andrade, C., Holgado, F., & Morales, F. J. (2018). Acrylamide content in French fries prepared in households: A pilot study in Spanish homes. Food Chemistry, 260, 44–52. Mesias, M., & Morales, F. J. (2016). Acrylamide in coffee: Estimation of exposure from vending machines. Journal of Food Composition and Analysis, 48, 8–12. Mishra, A. (2006). Production of L-asparaginase, an anticancer agent, from Aspergillus niger using agricultural waste in solid state fermentation. Applied Biochemistry and Biotechnology, 135, 33–42. Mohan Kumar, N. S., Shimray, C. A., Indrani, D., & Manonmani, H. K. (2014). Reduction of acrylamide formation in sweet bread with L-Asparaginase treatment. Food and Bioprocess Technology, 7, 741–748. Mottram, D. S., Wedzicha, B. L., & Dodson, A. (2002). Acrylamide is formed in the Maillard reaction. Nature, 419, 448–449. Mukherjee, J., Majumdar, T., & Scheper, T. (2000). Studies on nutritional and oxygen requirements for production of L-asparaginase by Enterobacter aerogenes. Applied Microbiology and Biotechnology, 53, 180–184. Muttucumaru, N., Powers, S. J., Elmore, J. S., Dodson, A., Briddon, A., ... Halford, N. G. (2017). Acrylamide-forming potential of potatoes grown at different locations, and the ratio of free asparagine to reducing sugars at which free asparagine becomes a limiting factor for acrylamide formation. Food Chemistry, 220, 76–86. Nguyen, H. T., van de Fels-Klerx, H. J., Peters, R. J. B., & van Boekel, M. A. J. S. (2016). Acrylamide and 5-hydroxymethylfurfural formation during baking of biscuits: Part I: Effects of sugar type. Food Chemistry, 192, 575–585. Patro, K., Basak, U., Mohapatra, A., & Gupta, N. (2014). Development of new medium composition for enhanced production of L-asparaginase by Aspergillus flavus. Journal of Environmental Biology, 35(1), 295–300. Patro, K., & Gupta, N. (2012). Extraction, purification and characterization of L-asparaginase from Penicillium sp. by submerged fermentation. International Journal for Biotechnology and Molecular Biology Research, 3, 30–34. Pedreschi, F. (2009). Acrylamide formation and reduction in fried potatoes. In E. Ortega-

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